Optimization of culture conditions to support undifferentiated growth of human embryonic stem cells.

Human embryonic stem cells (hESCs) are usually maintained in an undifferentiated state by coculture with mitomycin C-treated mouse embryonic fibroblasts (MEFs) as feeder cells in the presence of animal sera such as fetal bovine serum (FBS). Here, we use primary human amnion epithelial cells (hAECs) as feeder cells and human umbilical cord blood serum (CBS) as a replacement for FBS to support undifferentiated growth of hESCs. The 5 approximately 10-fold higher expression levels of ES cell markers including FGF, Oct-4, Nanog, Sox-2, Rex, and TERT were found in hESCs grown on hAECs compared with that on MEFs as measured by quantitative real-time polymerase chain reaction (PCR). By immunofluorescence, the expresisons of Oct-4 and Nanog is also higher in cells grown on hAECs than those on MEFs. Importantly, the ES cells grown on hAECs exhibit normal karyotypes on passage 25, thus ruling out the possibility of transformation. Using flow cytometry analysis, we show that both the ES cells grown on hAECs and MEFs have the same cell cycle distribution pattern. Further, hESCs cultured on hAECs for at least 20 passages could differentiate into three germ layers via teratoma formation. In addition, chromatin immunoprecipitation assay revealed that histone H3 is highly acetylated, and H3 lysine (K) 4 is hypermethylated at the Nanog locus and the Oct-4 locus in hESCs grown on hAECs. Conversely, hESCs grown on MEFs show histone deacetylation and H3-K4 demethylation. Taken together, these results suggest that hAECs supplemented with 10% CBS are suitable for hESC culture, and that this method may prove to be valuable for use in future regenerative therapies.